1. Field of the Invention
The invention relates to mutants and alleles of the prpD1 gene of coryneform bacteria coding for variants of 2-methylcitrate dehydratase (EC No. 4.2.1.79) and methods for producing amino acids, preferably L-lysine, L-tryptophan, L-valine, L-isoleucine and L-homoserine using bacteria which comprise these alleles.
2. Description of the Related Art
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.
Amino acids are used in human medicine, in the pharmaceutical industry, in the food product industry and in livestock nutrition.
Amino acids are produced by fermentation of strains of coryneform bacteria, for example, Corynebacterium glutamicum. Due to the great importance, work on improving the production methods is continually being done. Improvements in the methods may be fermentation technology measures such as, for example, stirring and supplying oxygen, or relate to the composition of the nutrient media, such as, for example, the sugar concentration during the fermentation, or the working up to the product form by for example ion exchange chromatography or the intrinsic output properties of the microorganism itself.
Methods used for improving the output properties of these microorganisms are ones of mutagenesis, selection and choice of mutants. The strains obtained in this way are resistant to antimetabolites or are auxotrophic for metabolites of regulatory importance, and produce the amino acids. A known antimetabolite is the lysine analogue S-(2-aminoethyl)-L-cysteine (AEC).
Methods of recombinant DNA technology have likewise been used for some years for strain improvement of L-amino acid-producing strains of Corynebacterium by amplifying individual amino acid biosynthesis genes and investigating the effect on amino acid production.
The chromosome of Corynebacterium glutamicum was completely sequenced some time ago (Kalinowski et al., Journal of Biotechnology 104, 5-25 (2003)). The chromosome of Corynebacterium efficiens has likewise been sequenced (Nishio et al., Genome Res. 13 (7), 1572-1579 (2003)).
Corresponding sequence data can be taken from the public databases. Suitable databases are for example the database of the European Molecular Biology Laboratories (EMBL, Heidelberg, Germany and Cambridge, UK), the database of the National Center for Biotechnology Information (NCBI, Bethesda, Md., USA), that of the Swiss Institute of Bioinformatics (Swissprot, Geneva, Switzerland), the Protein Information Resource Database (PIR, Washington, D.C., USA) and the DNA Data Bank of Japan (DDBJ, 1111 Yata, Mishima, 411-8540, Japan).
Summarizing descriptions of the genetics, the metabolism and the industrial importance of Corynebacterium are to be found in the articles by Ikeda, by Pfefferle et al. and by Mueller and Huebner in the book “Microbial Production of L-Amino Acids” (Advances in Biochemical Engineering 79, (2003), Springer Verlag, Berlin, Germany, editor: T. Scheper), in the special edition “A New Era in Corynebacterium glutamicum Biotechnology” of the Journal of Biotechnology (volume 104 (1-3), 2003, editors: A. Pühler and T. Tauch) and in the “Handbook of Corynebacterium glutamicum” (editors: L. Eggeling and M. Bott, CRC Press, Taylor & Francis Group, Boca Raton, Fla., USA, 2005).
The nucleotide sequence of the prpD1 gene coding for the 2-methylcitrate dehydratase of Corynebacterium glutamicum is available inter alia in the database of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, Md., USA) under the access number AF434798. It can furthermore be found in the Patent Application EP 1 108 790 as sequence No. 770.
Claes et al. (Journal of Bacteriology 184(10), 2728-2739 (2002)) report on genetic, microbiological and biochemical investigations on the prpD1, prpB1 and prpC1 genes of Corynebacterium glutamicum ATCC 13032.
For improved clarity, the nucleotide sequence of the prpD1 gene coding for the 2-methylcitrate dehydratase from Corynebacterium glutamicum (“wild-type gene”) according to the data of the NCBI database is depicted in SEQ ID NO:1, and the amino acid sequence of the encoded 2-methylcitrate dehydratase resulting therefrom is depicted in SEQ ID NO:2 and 4. Nucleotide sequences located upstream and downstream are additionally indicated in SEQ ID NO:3.